3-chloro-p-toluidine hydrochloride metabolism and detection of exposure in birds
Date
2017
Authors
Goldade, David A., author
Marie, Legare, advisor
William, Hannemann, advisor
Gregory, Dooley, committee member
Gary, Mason, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
The avicide 3-chloro-4-methylanaline hydrochloride (chloro-p-toluidine hydrochloride, CPTH, DRC-1339) is used to control pest bird species that damage agricultural crops. While it is theorized that CPTH is a nephrotoxin, no definitive assessment of the mode of action has been performed. Additionally, the metabolic pathway of CPTH in birds has yet to be elucidated. Radioactively labeled [14C]-3-chloro-4-methylaniline hydrochloride (250 µg per bird) was delivered to 21 red-winged blackbirds (Agelaius phoeniceus) and 21 dark-eyed juncos (Junco hyemalis) via oral gavage, and the distribution and excretion of radioactivity were determined at 15 and 30 minutes and 1, 4, 8, 12, and 24 hours (n = 3 per time point). Direct measurement of radioactivity as well as measurement following combustion was accomplished using a liquid scintillation counter. Elimination from most tissues followed a two-compartment model, with very rapid elimination occurring between time 0 and 4 hours and a much slower elimination phase occurring after that. The average half-life of elimination for the initial phase in most tissues examined was 0.16 hours for juncos and 0.62 hours for blackbirds. The average for the slower second phase of elimination was 3.4 hours for juncos and 5.4 hours for blackbirds. The radioactivity in blackbird kidney tissues did not change significantly for the duration of the test, pointing toward the kidney as a possible site of action for this important agricultural chemical. To further explore the mechanistic toxicology of CPTH, sub-cellular preparations were made from the liver and kidney of various avian species. In-vitro metabolism experiments were performed using these preparations and the resulting metabolites were identified and quantified. Two metabolites were identified: 3-chloro-4-methylacetanilide (CAT) and N-[3-chloro-4-(hydroxymethyl)phenyl]acetamide (OH-CAT). A comparison of two methods was made for the analysis of CPTH and its metabolites. Due primarily to the solubility and volatility of the three compounds, CPTH and CAT performed well on gas chromatography tandem mass spectrometry (GC/MS/MS) and adequately on liquid chromatography tandem mass spectrometry (LC/MS/MS). Conversely, OH-CAT performed optimally on LC/MS/MS. LC/MS/MS was chosen as the technique for analysis of exposure data. Both methods generated residue values that demonstrated a high degree of variability between individuals. Despite the variability issues, the data showed that the primary chemical species present in the tissues of exposed birds was OH-CAT, and that the concentration of observed residue was related to the dose administered. In an effort to identify the target for tissue binding of CPTH or its metabolites in the kidney of exposed red-winged blackbirds, protein samples were extracted and digested with trypsin. Several chemical compounds were found to be significantly different between treated and control groups (α=0.05) and were subjected to tandem mass spectrometry to identify their chemical structure. Results from this analysis did not yield any identification of specific protein binding. Limitations of sensitivity and lack of sample enrichment likely led to this outcome.